Bottom Line:
The naked 14-nt sgRNA significantly reduced the miR-16 level in HEK293 and HL60 cells.Three other naked 14-nt sgRNAs against miR-142-3p, miR-206, and miR-19a/b are also shown to downregulate the respective miRNA levels in various mammalian cell lines.Our observations suggest that in general we can eliminate a specific cellular miRNA at least by ~50% by using a naked 14-nt sgRNA on the basis of TRUE gene silencing.

Affiliation: Department of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata, Japan.

ABSTRACTtRNase Z(L)-utilizing efficacious gene silencing (TRUE gene silencing) is a newly developed technology to suppress mammalian gene expression. TRUE gene silencing works on the basis of a unique enzymatic property of mammalian tRNase Z(L), which is that it can recognize a pre-tRNA-like or micro-pre-tRNA-like complex formed between target RNA and artificial small guide RNA (sgRNA) and can cleave any target RNA at any desired site. There are four types of sgRNA, 5'-half-tRNA, RNA heptamer, hook RNA, and ~14-nt linear RNA. Here we show that a 14-nt linear-type sgRNA against human miR-16 can guide tRNase Z(L) cleavage of miR-16 in vitro and can downregulate the miR-16 level in HEK293 cells. We also demonstrate that the 14-nt sgRNA can be efficiently taken up without any transfection reagents by living cells and can exist stably in there for at least 24 hours. The naked 14-nt sgRNA significantly reduced the miR-16 level in HEK293 and HL60 cells. Three other naked 14-nt sgRNAs against miR-142-3p, miR-206, and miR-19a/b are also shown to downregulate the respective miRNA levels in various mammalian cell lines. Our observations suggest that in general we can eliminate a specific cellular miRNA at least by ~50% by using a naked 14-nt sgRNA on the basis of TRUE gene silencing.

pone-0038496-g005: Downregulation of miRNA expression levels by naked 14-nt sgRNAs.(A) Real-time PCR analyses for miR-142-3p were carried out for total RNAs extracted from HL60 and DAUDI cells that were cultured for 24 hours in the absence or presence of sgR142(1–14) or sgR142(1–23). The miR-142-3p levels are normalized against the RNU48 levels. (B) Real-time PCR analysis for miR-206 was performed for total RNA extracted from C2C12 cells that were cultured for 24 hours in the absence or presence of sgR206(1–14). The miR-206 levels are normalized against the sno234 RNA levels. (C) Real-time PCR analysis for miR-19a/b was performed for total RNA extracted from RPMI-8226 cells that were cultured for 24 hours in the absence or presence of sgR19(1–14). The miR-19a/b levels are normalized against the 5S rRNA levels. Error bars indicate s.d. (n = 3). *, P<0.05; **, P<0.001.

Mentions:
We also examined three naked 14-nt sgRNAs against miR-142-3p, miR-206, and miR-19a/b for their guiding ability. sgR142(1–14) significantly reduced the miR-142-3p level to 48% and 47% in HL60 and DAUDI cells, respectively, without any transfection reagents, whereas sgR142(1–23) decreased the level slightly in both cell lines (Figure 5A). The 22-nt 5′- and 3′-phosphorylated sgR16(1–22) worked very efficiently even in a naked form (Figure 4B), whereas the 23-nt sgR142(1–23), which was also 5′- and 3′-phosphorylated, barely worked, suggesting that, unlike 14-nt sgRNAs, the efficiency of naked 22–23-nt RNAs in being taken up by living cells and/or their stability in the cells may change depending on their sequence itself as well as 3′-phosphorylation status.

pone-0038496-g005: Downregulation of miRNA expression levels by naked 14-nt sgRNAs.(A) Real-time PCR analyses for miR-142-3p were carried out for total RNAs extracted from HL60 and DAUDI cells that were cultured for 24 hours in the absence or presence of sgR142(1–14) or sgR142(1–23). The miR-142-3p levels are normalized against the RNU48 levels. (B) Real-time PCR analysis for miR-206 was performed for total RNA extracted from C2C12 cells that were cultured for 24 hours in the absence or presence of sgR206(1–14). The miR-206 levels are normalized against the sno234 RNA levels. (C) Real-time PCR analysis for miR-19a/b was performed for total RNA extracted from RPMI-8226 cells that were cultured for 24 hours in the absence or presence of sgR19(1–14). The miR-19a/b levels are normalized against the 5S rRNA levels. Error bars indicate s.d. (n = 3). *, P<0.05; **, P<0.001.

Mentions:
We also examined three naked 14-nt sgRNAs against miR-142-3p, miR-206, and miR-19a/b for their guiding ability. sgR142(1–14) significantly reduced the miR-142-3p level to 48% and 47% in HL60 and DAUDI cells, respectively, without any transfection reagents, whereas sgR142(1–23) decreased the level slightly in both cell lines (Figure 5A). The 22-nt 5′- and 3′-phosphorylated sgR16(1–22) worked very efficiently even in a naked form (Figure 4B), whereas the 23-nt sgR142(1–23), which was also 5′- and 3′-phosphorylated, barely worked, suggesting that, unlike 14-nt sgRNAs, the efficiency of naked 22–23-nt RNAs in being taken up by living cells and/or their stability in the cells may change depending on their sequence itself as well as 3′-phosphorylation status.

Bottom Line:
The naked 14-nt sgRNA significantly reduced the miR-16 level in HEK293 and HL60 cells.Three other naked 14-nt sgRNAs against miR-142-3p, miR-206, and miR-19a/b are also shown to downregulate the respective miRNA levels in various mammalian cell lines.Our observations suggest that in general we can eliminate a specific cellular miRNA at least by ~50% by using a naked 14-nt sgRNA on the basis of TRUE gene silencing.

Affiliation:
Department of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata, Japan.

ABSTRACTtRNase Z(L)-utilizing efficacious gene silencing (TRUE gene silencing) is a newly developed technology to suppress mammalian gene expression. TRUE gene silencing works on the basis of a unique enzymatic property of mammalian tRNase Z(L), which is that it can recognize a pre-tRNA-like or micro-pre-tRNA-like complex formed between target RNA and artificial small guide RNA (sgRNA) and can cleave any target RNA at any desired site. There are four types of sgRNA, 5'-half-tRNA, RNA heptamer, hook RNA, and ~14-nt linear RNA. Here we show that a 14-nt linear-type sgRNA against human miR-16 can guide tRNase Z(L) cleavage of miR-16 in vitro and can downregulate the miR-16 level in HEK293 cells. We also demonstrate that the 14-nt sgRNA can be efficiently taken up without any transfection reagents by living cells and can exist stably in there for at least 24 hours. The naked 14-nt sgRNA significantly reduced the miR-16 level in HEK293 and HL60 cells. Three other naked 14-nt sgRNAs against miR-142-3p, miR-206, and miR-19a/b are also shown to downregulate the respective miRNA levels in various mammalian cell lines. Our observations suggest that in general we can eliminate a specific cellular miRNA at least by ~50% by using a naked 14-nt sgRNA on the basis of TRUE gene silencing.